Phosphorylation of cyclin O, a novel cyclin family protein containing a cyclin-like domain, is involved in the activation of cyclin-dependent kinase 2.

Kim DH, Park JH, Lee B, Jang KO, Chung IS, Han YS - Oncol Lett (2014)

Bottom Line:
Treatment with calf intestinal phosphatase reduced the intensity of the uppermost band.This activity was reduced in cells overexpressing cyclin O, in which the 81st serine had been replaced with alanine (S81A).These results suggest that cyclin O is a novel cyclin family protein that regulates CDK2 kinase activity, which is mediated by the phosphorylation of the 81st serine residue of cyclin O.

ABSTRACTCell cycles, ordered series of events modulating cell growth and division, are tightly regulated by complexes containing cyclin-dependent kinases (CDKs) and cyclins. Cyclin O is a novel cyclin family protein which interacts with CDK2. However, the molecular effects of cyclin O on the activity of CDK2 have not been fully evaluated. In this study, an interaction between cyclin O and CDK2 was identified by co-immunoprecipitation and the effect of cyclin O on the kinase activity of CDK2 was investigated using cyclin O point mutants. Co-immunoprecipitation was achieved using using HEK293 human embryonic kidney cells which were transiently transfected with vectors expressing cyclin O and CDK2, which revealed that cyclin O interacted with CDK2, particularly with the active form of endogenous CDK2. Cyclin O was expressed as several different bands with molecular weights between 45 and 50 kDa, possibly due to different post-translational modifications. When co-expressed with CDK2, cyclin O appeared as a band with a molecular weight of 50 kDa. Treatment with calf intestinal phosphatase reduced the intensity of the uppermost band. Mass spectroscopic analysis of cyclin O co-expressed with CDK2 revealed that the 81st serine residue of cyclin O was phosphorylated. The in vitro kinase activity of CDK2 phosphorylating histone H1 was markedly increased in the cells overexpressing cyclin O. This activity was reduced in cells overexpressing cyclin O, in which the 81st serine had been replaced with alanine (S81A). These results suggest that cyclin O is a novel cyclin family protein that regulates CDK2 kinase activity, which is mediated by the phosphorylation of the 81st serine residue of cyclin O.

Mentions:
To assess the in vitro kinase activity of CDK2, HEK 293 cell lysates were immunoprecipitated with anti-CDK2 or rabbit IgG as a control. CDK2 was immunoprecipitated by anti-CDK2, but not rabbit IgG (Fig. 5A). The immunoprecipitates were incubated with HH1 in the presence of [γ-32P] ATP. [γ-32P]-incorporated HH1 was detected in the anti-CDK2 immunoprecipitate reaction mixture (Fig. 5B, lane 5), but not in the rabbit IgG reaction mixture (Fig. 5B, lane 4). The cell lysates obtained from the HEK 293 cells transiently transfected with vectors expressing c-myc-CyO or c-myc-CyO S81A were immunoprecipitated with anti-CDK2, and a kinase activity assay was performed. The CDK2 kinase activities of the immunoprecipitates obtained from the HEK 293 cells that were transiently transfected with vectors expressing c-myc-CyO and c-myc-CyO S81A were increased by 2.5- and 1.6-fold, respectively, compared with those of the HEK 293 cells transiently transfected with the empty vector, pCMV Tag3A (Fig. 5C and D).

Mentions:
To assess the in vitro kinase activity of CDK2, HEK 293 cell lysates were immunoprecipitated with anti-CDK2 or rabbit IgG as a control. CDK2 was immunoprecipitated by anti-CDK2, but not rabbit IgG (Fig. 5A). The immunoprecipitates were incubated with HH1 in the presence of [γ-32P] ATP. [γ-32P]-incorporated HH1 was detected in the anti-CDK2 immunoprecipitate reaction mixture (Fig. 5B, lane 5), but not in the rabbit IgG reaction mixture (Fig. 5B, lane 4). The cell lysates obtained from the HEK 293 cells transiently transfected with vectors expressing c-myc-CyO or c-myc-CyO S81A were immunoprecipitated with anti-CDK2, and a kinase activity assay was performed. The CDK2 kinase activities of the immunoprecipitates obtained from the HEK 293 cells that were transiently transfected with vectors expressing c-myc-CyO and c-myc-CyO S81A were increased by 2.5- and 1.6-fold, respectively, compared with those of the HEK 293 cells transiently transfected with the empty vector, pCMV Tag3A (Fig. 5C and D).

Bottom Line:
Treatment with calf intestinal phosphatase reduced the intensity of the uppermost band.This activity was reduced in cells overexpressing cyclin O, in which the 81st serine had been replaced with alanine (S81A).These results suggest that cyclin O is a novel cyclin family protein that regulates CDK2 kinase activity, which is mediated by the phosphorylation of the 81st serine residue of cyclin O.

ABSTRACTCell cycles, ordered series of events modulating cell growth and division, are tightly regulated by complexes containing cyclin-dependent kinases (CDKs) and cyclins. Cyclin O is a novel cyclin family protein which interacts with CDK2. However, the molecular effects of cyclin O on the activity of CDK2 have not been fully evaluated. In this study, an interaction between cyclin O and CDK2 was identified by co-immunoprecipitation and the effect of cyclin O on the kinase activity of CDK2 was investigated using cyclin O point mutants. Co-immunoprecipitation was achieved using using HEK293 human embryonic kidney cells which were transiently transfected with vectors expressing cyclin O and CDK2, which revealed that cyclin O interacted with CDK2, particularly with the active form of endogenous CDK2. Cyclin O was expressed as several different bands with molecular weights between 45 and 50 kDa, possibly due to different post-translational modifications. When co-expressed with CDK2, cyclin O appeared as a band with a molecular weight of 50 kDa. Treatment with calf intestinal phosphatase reduced the intensity of the uppermost band. Mass spectroscopic analysis of cyclin O co-expressed with CDK2 revealed that the 81st serine residue of cyclin O was phosphorylated. The in vitro kinase activity of CDK2 phosphorylating histone H1 was markedly increased in the cells overexpressing cyclin O. This activity was reduced in cells overexpressing cyclin O, in which the 81st serine had been replaced with alanine (S81A). These results suggest that cyclin O is a novel cyclin family protein that regulates CDK2 kinase activity, which is mediated by the phosphorylation of the 81st serine residue of cyclin O.